Detection of specific nucleic acid sequences present in cells is generally known in the art. Southern (J. Mol. Biol. (1975) 98: 503-517) teaches the detection of specific sequences among DNA fragments separated by gel electrophoresis using "blotting" or transfer of the DNA fragments to a membrane, followed by hybridization of denatured DNA fragments with a radioactive probe and autoradiography. This procedure has also been extended to the detection of RNA molecules extracted from cells or tissues. More recently, faster and quantitative "dot-blotting" procedures have been developed for rapid detection of DNA or RNA from tissues or cells.
Recently, considerable interest has been generated in the development of synthetic oligonucleotides as therapeutic or gene expression modulating agents in the so-called antisense approach. These agents, called antisense oligonucleotides, bind to a target single-stranded nucleic acid molecule according to the Watson-Crick or the Hoogstein rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal translation or transcription; in the case of an mRNA target, by triggering the enzymatic destruction of the messenger by RNase H; or by destroying the target via reactive groups attached directly to the antisense oligonucleotide.
Antisense oligodeoxynucleotides have been designed to specifically inhibit the expression of HIV-1 and other viruses (see, e.g., Agrawal (1992) Trends in Biotechnology 10: 152-158; Agrawal et al. in Gene Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds.) Raven Press Ltd., New York (1992) pp. 273-283); Matsukura et al. in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, Wiley-Liss, Inc. (1992) pp. 159-178; and Agrawal (1991) in Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS, (Wickstrom, ed. ) Liss, New York, pp. 145-148). For example, it has been shown that antisense oligonucleotides having unmodified phosphodiester or modified internucleoside bonds and sequences complementary to portions of genomic HIV-1 ribonucleic acid (RNA) inhibit viral replication in early infected cells (Zamecnik et al. (1986) Proc. Natl. Acad. Sci. (USA) 83: 4143-4147; Goodchild et al. (1988) Proc. Natl. Acad. Sci (USA) 85: 5507-5511).
However, molecules with unmodified phosphodiester bonds are less able to inhibit viral replication in chronically infected cells (Agrawal et al. (1989) Proc. Natl. Acad. Sci. (USA) 86: 7790-7794), mainly because of their nuclease susceptibility (Wickstrom (1986) J. Biochem. Biophys. Meth. 13: 97-102). Therefore, chemically modified, nuclease-resistant analogs have been developed which are effective in inhibiting HIV-1 replication in tissue cultures (Sarin et al. (1988) Proc. Natl Acad. Sci. (USA) 85: 7448-7451; Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85: 7079-7083; Matsukura et al. (1988) Gene 72: 343-347). These analogs include oligonucleotides with nuclease-resistant phosphorothioate internucleotide linkages shown to inhibit HIV-1 replication in both acute infection (Agrawal et al. (1989) Proc. Natl. Acad. Sci (USA) 86: 7790-7794) and in chronically infected cell lines (Agrawal et al. (1991) in Gene Regulation: Biology of Antisense RNA, (Erickson et al., eds.) Raven Press, New York, pp. 273-284; Vickers et al. (1991) Nucleic Acids Res. 19: 3359-3368; Matsukura et al. (1989) Proc. Natl. Acad. Sci. (USA) 86: 4244-4248; Agrawal et al. (1988) Proc. Natl Acad. Sci. (USA) 85: 7079-7083).
For an antisense therapeutic approach to be effective, oligonucleotides must be introduced into a subject and must reach the specific tissues to be treated. Consequently, analytical methods are needed to detect oligonucleotides in body fluids or tissues.
Temsamani et al. (U.S. patent application Ser. No. 08/002,786) developed a method of extracting oligonucleotides which had been proteolytically digested from body fluid or tissue samples. Total nucleic acids are precipitated from the extracted samples and transferred to a hybridization membrane where they are hybridized to a labelled oligonucleotide that is complementary to the oligonucleotide that was administered to the subject. Presence of the hybridized, labelled oligonucleotide is then detected by standard procedures.
Radiolabelled oligonucleotides have been administered to animal models and their distribution within body fluids and tissues has been assessed by extraction of the oligonucleotides followed by autoradiography (see Agrawal et al. (1991) Proc. Natl. Acad. Sci. (USA) 88: 7595-7599). As a practical matter, however, these methods have not been exercised in human patients.
Unfortunately, the various techniques for detecting specific unlabelled nucleic acid sequences present in body fluids or tissues has thus far only been extended to polynucleotides such as large DNA or RNA molecules. Due to the small size of antisense oligonucleotides, special problems relating to nonspecific binding or background, as well as to absence of binding, nondetection, or false negatives exist. Thus, there remains a need to develop procedures for the detection of specific synthetic oligonucleotide sequences present in biological fluids such as body fluids and tissues.
Bioavailability and pharmacokinetic measurements of metabolites in samples of blood, tissue, urine, and other biological fluids, have been made using detection methods involving autoradiography (Agrawal et al. (1991) Proc. Natl. Acad. Sci. (USA) 88: 7595). In addition, detection of oligonucleotides has been accomplished by UV detection of a natural chromophore in the molecule. However, quantitative detection of oligonucleotides by UV is limited to concentrations of 50-100 parts per billion (ppb) or about 10.sup.-8 M in the sample vials at best. When more sensitive detection is required, radioactive and laser induced fluorescence (LIF) are the methods of choice. For example, laser-induced fluorescence has the capacity to detect small numbers of rhodamine molecules in an aqueous flowing solution (Dovichi et al. (1984) Anal. Chem. 56: 348-354).
Since neither radioactive nor fluorescent labelled oligonucleotides are used in humans, direct determination of oligonucleotide analogs in biological fluids at very low concentrations (sub-ppb) is currently impossible. A combination of capillary gel electrophoresis and laser-induced fluorescence has been used to measure DNA sequence reaction products (see, e.g., Cohen et al. (1990) J. Chromatogr. 516: 49-60; Swerdlow et al. (1990) Nucl. Acids Res. 18: 1415-1418) and derivatized amino acids (Cheng et al. (1988) Science 242: 562-564). However, these methods are unable to detect minute levels of small oligonucleotides such as those useful in antisense therapy which are in their native form.
Thus, at present there remains a need for methods of detecting and quantitating very low levels of small oligonucleotides in biological fluids.